JP2011104572A - Method for manufacturing liposome and flow manufacturing apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for manufacturing liposome, capable of enhancing productivity of good liposome. <P>SOLUTION: The method is a method for continuously manufacturing liposome, and includes a primary emulsion process for preparing W1/O emulsion; a secondary emulsion process for preparing W1/O/W2 emulsion; and a solvent removal process for removing an organic solvent of an oil phase (O) from the W1/O/W2 emulsion to form liposome. The solvent removal process receives continuous supply of the W1/O/W2 emulsion prepared in the secondary emulsion process, for example, by introducing the W1/O/W2 emulsion into a flow path for solvent removal and evaporating the organic solvent while falling it down for manufacture of liposome by the time when it reaches a downstream flow path terminal. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a method and apparatus for producing liposomes having uses such as pharmaceuticals, cosmetics and foods.

A liposome is a closed endoplasmic reticulum composed of a lipid bilayer mainly composed of phospholipid. Since it has a structure and function similar to a cell membrane, it is difficult to stimulate the immune system (low antigen) and is highly safe as a material. In addition, water-soluble drugs can be incorporated into the inner aqueous phase surrounded by lipid bilayer membranes, and fat-soluble drugs can be incorporated into lipid bilayer membranes. Can be retained. Therefore, research and development of efficient manufacturing methods and equipment for liposomes used in the fields of pharmaceuticals, cosmetics, foods, etc., for example, liposome preparations suitable for use in drug delivery systems (DDS) Is underway.

  Conventionally, a primary emulsification step in which an inner aqueous phase (W1) and an oil phase (O) are emulsified using a mixed lipid component (F1) for liposomes to prepare a W1 / O emulsion, and a primary emulsification step by a membrane emulsification method. The W1 / O emulsion and the external aqueous phase (W2) were emulsified with the mixed lipid component (F2) for liposomes to prepare a W1 / O / W2 emulsion, and a secondary emulsification step and a secondary emulsification step. A method for producing liposomes is known, which includes a solvent removal step of removing the organic solvent of the oil phase (O) from the W1 / O / W2 emulsion to form liposomes. In the solvent removal step, a method (in-liquid drying method) is known in which the W1 / O / W2 emulsion is transferred into an open container and the solvent is removed under reduced pressure and / or stirring.

  However, conventional solvent removal methods such as in-liquid drying methods tend to cause contact and aggregation between liquid enemies in the W1 / O / W2 emulsion, leading to a reduction in liposome quality. In order to prevent such contact and aggregation, the W1 / O / W2 emulsion can be added with a predetermined dispersion stabilizer. However, the dispersion stabilizer is not mixed into the final product, particularly a pharmaceutical liposome preparation. Undesirably, additional steps (such as ultrafiltration) are required to remove the dispersion stabilizer. For these reasons, conventional liposome production methods do not necessarily have high productivity of high-quality liposomes, and there is room for improvement.

  By the way, Patent Document 1 discloses an apparatus for producing a monodispersed composite emulsion (W / O / W emulsion) in which the dispersed phase itself constituting the emulsion is another emulsion, and this apparatus has a dispersed phase. A first space to be supplied; a second space adjacent to the first space and supplied with a continuous phase; and a second space adjacent to the second space and supplied with the same or different continuous phase as the continuous phase. 3 spaces, wherein the first space and the second space are connected by a first microchannel, and the second space and the third space are connected by a second microchannel. An apparatus is described (claim 1).

Patent Document 2 includes (a) a step of providing a mixing chamber and a solvent removing unit; (b) (i)
Continuously supplying a first emulsion to a mixing chamber, comprising: an organic solution comprising a polymeric material; and (ii) an organic solvent mixed with a first aqueous solution containing nucleic acid; (c) a surfactant; Continuously supplying a second aqueous solution containing the mixture into the mixing chamber; (d) continuously emulsifying the first emulsion and the second aqueous solution in the mixing chamber to form a second emulsion, A second emulsion comprising nucleic acid, polymeric material, water, and organic solvent; (e) continuously transferring the second emulsion from the mixing chamber to the solvent removal section; and (f) the solvent. A step of removing an organic solvent from the second emulsion in the removing section to form an aqueous dispersion of nucleic acid-containing fine particles, wherein at least one of the first emulsion and the second aqueous solution further comprises a dispersion stabilizer. A scalable continuous process for the preparation of inclusion microparticles is described (claim 1). In addition, it is described that using a homogenizer as the mixing chamber uses evaporation, heating, partial vacuum, or the like to remove the organic solvent (claims 17, 20, 21, 24).
However, it is described in both Patent Documents 1 and 2 that the above-described apparatus or method is applied to the production of liposomes formed with lipid bilayer membranes and having an average particle size of about several hundred nm. Not.

JP 2003-71261 A JP-T-2003-514832

  An object of this invention is to provide the manufacturing method and manufacturing apparatus which can improve the productivity of a quality liposome. In particular, in the step of removing the solvent from the oil phase of the W1 / O / W2 emulsion to form liposomes, a method for producing liposomes capable of suppressing contact and aggregation between droplets without using a complicated method, and An object is to provide a manufacturing apparatus.

  In order to complete the present invention, the present inventors have found that it is extremely important to solve the above-mentioned problems particularly in efficiently performing the procedure from the secondary emulsification step to the solvent removal step. It came.

  That is, the continuous production method of the liposome according to the present invention comprises primary emulsification in which an inner aqueous phase (W1) and an oil phase (O) are emulsified using the mixed lipid component (F1) for liposome to prepare a W1 / O emulsion. A secondary emulsification step of emulsifying the W1 / O emulsion prepared in the primary emulsification step and the outer aqueous phase (W2) using the mixed lipid component for liposome (F2) to prepare a W1 / O / W2 emulsion; A method for producing liposomes, comprising a solvent removal step of removing the organic solvent of the oil phase (O) from the W1 / O / W2 emulsion prepared in the secondary emulsification step to form liposomes, the solvent removal step comprising: It is characterized in that liposomes are continuously produced by receiving a continuous supply of the W1 / O / W2 emulsion prepared in the secondary emulsification step.

  In such a production method, the continuous production of liposomes in the solvent removal step is performed by introducing the W1 / O / W2 emulsion into the solvent removal flow path, evaporating the organic solvent while flowing down the solvent removal flow path, It is preferable to be achieved by forming liposomes until reaching the end.

  The continuous supply of the W1 / O / W2 emulsion prepared in the secondary emulsification step to the solvent removal step is performed by performing the solvent removal step under reduced pressure, and further in the solvent removal step. It is preferable that the pressure is controlled so as to be performed at a constant speed based on the result of detecting the degree of decompression and the liquid feeding pressure of the W1 / O / W2 emulsion. The solvent removal step is preferably placed under a reduced pressure of 750 to 180 mmHg.

As the emulsification method in the secondary emulsification step, for example, a membrane emulsification method is preferable, and an emulsification method using a microchannel emulsification method or an SPG membrane is particularly preferable. On the other hand, as an emulsification method in the primary emulsification step, for example, a stirring emulsification method is preferable.

  The primary emulsification step may be performed after adding a substance to be included in the liposome. Further, the outer aqueous phase (W2) in the secondary emulsification step forms a dispersion stabilizer, for example, a dispersion stabilizer that does not form a molecular assembly by itself or a molecular assembly by itself, but the volume average of the molecular assembly. You may contain the dispersion stabilizer whose particle size is 10 nm or less.

  Moreover, this invention provides the manufacturing apparatus of the liposome provided with the means which enforces the manufacturing method of this invention as mentioned above in one side surface.

  That is, the continuous production apparatus for liposome according to the present invention comprises primary emulsification in which an inner aqueous phase (W1) and an oil phase (O) are emulsified using a mixed lipid component (F1) for liposomes to prepare a W1 / O emulsion. And a secondary emulsifying part for emulsifying the W1 / O emulsion prepared in the primary emulsifying part and the outer aqueous phase (W2) using the mixed lipid component for liposome (F2) to prepare a W1 / O / W2 emulsion, , An apparatus for producing liposomes comprising a solvent removal unit for removing the organic solvent of the oil phase (O) from the W1 / O / W2 emulsion prepared in the secondary emulsification unit to form liposomes, wherein the solvent removal unit Is characterized in that the liposome is continuously produced by receiving a continuous supply of the W1 / O / W2 emulsion prepared in the secondary emulsification unit.

  Such a production apparatus preferably includes a solvent removal flow path for evaporating the organic solvent while introducing and flowing the W1 / O / W2 emulsion for continuous production of liposomes in the solvent removal unit.

  The solvent removal unit preferably includes a decompression device, and the decompression device is used to continuously supply the W1 / O / W2 emulsion prepared in the secondary emulsification unit to the solvent removal unit. .

  Furthermore, the above-described manufacturing apparatus is provided with a degree of pressure reduction in the solvent removal step and a W1 / O / W2 emulsion feed for continuous supply to the solvent removal step of the W1 / O / W2 emulsion prepared in the secondary emulsification step. It is preferable to provide a supply control means including a member for detecting the liquid pressure and a member for controlling the supply speed to be constant based on the detection result.

  As an emulsification method in the secondary emulsification part, for example, a membrane emulsification method is preferable, and an emulsification method using a microchannel emulsification method or an SPG membrane is particularly preferable. On the other hand, as an emulsification method in the primary emulsification part, for example, a stirring emulsification method is preferable.

  According to the liposome production method and production apparatus of the present invention, since liposomes can be continuously produced while suppressing contact and aggregation of droplets in the W1 / O / W2 emulsion, the encapsulation rate is high, Productivity of high-quality liposomes with uniform particle size distribution (small CV value) is remarkably improved, and industrialization can be realized. In particular, in a preferred embodiment of the present invention, when the reduced pressure of the solvent removal step and the solvent removal apparatus is utilized, the effects such as the improvement in productivity as described above can be further enjoyed. Moreover, aggregation can be suppressed to a certain extent without using a dispersion stabilizer, and aggregation can be more reliably prevented by using a dispersion stabilizer. Furthermore, since the primary emulsification step, the secondary emulsification step and the solvent removal step can all be carried out in a closed system, it is possible to prevent foreign substances from being mixed and to produce highly safe liposomes (particularly liposome preparations for pharmaceutical use). it can.

FIG. 1 is a schematic view of one embodiment of the liposome production apparatus of the present invention. FIG. 2 is a microscopic photograph of the liposome suspension prepared in Example 1. FIG. 3 is a microscopic photograph of the liposome suspension prepared in Example 2. 4 is a microscopic observation photograph of the liposome suspension prepared in Comparative Example 1. FIG. FIG. 5 is a microscopic photograph of the liposome suspension prepared in Comparative Example 2.

− Method for producing liposome −
The method for producing liposomes of the present invention has the following primary emulsification step, secondary emulsification step and solvent removal step, and can be appropriately combined with other steps as necessary.

(Primary emulsification process)
The primary emulsification step is a step of preparing a W1 / O emulsion by emulsifying the inner aqueous phase (W1) and the oil phase (O) using the mixed lipid component (F1) for liposomes.

  As a method for preparing the W1 / O emulsion, known methods such as an ultrasonic emulsification method, a stirring emulsification method, a membrane emulsification method, and a method using a high-pressure homogenizer can be applied. A membrane emulsification method is preferable from the viewpoint of supplying to the secondary emulsification step, a high-pressure homogenizer is preferable from the viewpoint of fine particle size, and a stirring emulsification method is preferable from the viewpoint of scale-up. As a membrane emulsification method, a premix membrane emulsification method is used in which a W1 / O emulsion having a large particle size is prepared in advance and then a W1 / O emulsion having a smaller particle size is prepared by passing through a membrane having a small pore size. May be.

  In the primary emulsification step, the average particle size of the W1 / O emulsion, the ratio of the mixed lipid component (F1) added to the oil phase (O), the volume ratio of the oil phase (O) to the inner aqueous phase (W1), and other conditions Can be appropriately adjusted according to the emulsification method to be employed, taking into consideration the conditions of the subsequent secondary emulsification step and the aspect of the liposome to be finally prepared. Usually, the ratio of the mixed lipid component (F1) is 1 to 50% by mass with respect to the oil phase (O), and the volume ratio of the oil phase (O) to the inner aqueous phase (W1) is 100: 1 to 1: 2. It is. Further, if necessary, substances (water-soluble or fat-soluble drugs) to be encapsulated in liposomes are added in the primary emulsification step, and dissolved or suspended in the inner aqueous phase (W1) or the oil phase (O). You may leave it.

  After the primary emulsification step, the prepared W1 / O emulsion is fed to the secondary emulsification step while being synchronized with the continuous preparation of the W1 / O / W2 emulsion in the secondary emulsification step.

(Secondary emulsification process)
The secondary emulsification step is a step of emulsifying the W1 / O emulsion and the external aqueous phase (W2) prepared in the primary emulsification step using the mixed lipid component (F2) for liposome to prepare a W1 / O / W2 emulsion. is there. In this secondary emulsification step, the W1 / O / W2 emulsion is continuously fed to the solvent removal step, and the W1 / O emulsion prepared in the primary emulsification step is continuously fed while the W1 / O / W2 emulsion is continuously fed. An O / W2 emulsion is prepared continuously.

As a method for preparing the W1 / O / W2 emulsion in the secondary emulsification step, a membrane emulsification method, a stirring emulsification method, a droplet method, a contact method, and the like are known, but the W1 / O / W2 emulsion is continuously used. In the present invention, it is preferable to use the membrane emulsification method from the viewpoint of preparing the solution and supplying it to the solvent removal step. Among the membrane emulsification methods, the microchannel emulsification method and the emulsification method using the SPG membrane do not require a large mechanical shearing force for the emulsification process, and therefore, the droplet collapse during the emulsification operation and the inclusion material from the droplets Leakage can be suppressed, and droplets are released into the continuous phase that flows down and are transported to the solvent removal step sequentially without staying, reducing contact and aggregation between the droplets and suppressing formation of multivesicular liposomes. This is preferable. As a membrane emulsification method, a W1 / O / W2 emulsion having a large particle size is prepared in advance, and then a W1 / O / W2 emulsion having a smaller particle size is prepared by passing through a membrane having a small pore size. A mixed film emulsification method may be used. The premix membrane emulsification method is preferable because it requires a small amount of energy, requires a large amount of treatment, and can speed up the preparation of liposomes.

  In the secondary emulsification step, conditions such as the formation speed or density of the droplets in the W1 / O / W2 emulsion and the flow rate of the continuous phase reduce the probability of the droplets contacting and aggregating, and liposomes in the solvent removal step The W1 / O / W2 emulsion can be appropriately adjusted so as to be produced at a predetermined speed synchronized with the production of As needed, you may mix | blend a dispersion stabilizer with the outer water phase (W2) of a secondary emulsification process. Moreover, what is necessary is just to adjust suitably about other conditions, such as the addition amount of a mixed lipid component (F2), considering the use etc. of the liposome finally prepared.

  In addition, when the mixed lipid component (F2) for emulsification in the secondary emulsification step is mainly composed of a water-soluble lipid, it is used by being added to the outer aqueous phase (W2), but when it is mainly composed of a fat-soluble lipid, Since it adds and uses for the prepared W1 / O emulsion, you may add the addition process for it as needed. In addition, when the mixed lipid components (F1) and (F2) may have the same composition, the surplus that could not be fully oriented at the W / O interface as F1 among the mixed lipid components added during the primary emulsification step The minute can be F2 to be oriented at the O / W interface of the secondary emulsification step.

  After the secondary emulsification step, the prepared W1 / O / W2 emulsion is continuously discharged from the secondary emulsification step and synchronized with the solvent removal step while synchronizing with the continuous preparation of liposomes in the solvent removal step. To be supplied. At this time, the continuous supply of the W1 / O / W2 emulsion to the solvent removal step is, for example, based on the result of detecting the degree of pressure reduction in the solvent removal step and the liquid feeding pressure of the W1 / O / W2 emulsion. It is desirable to be controlled to be done at speed. For example, if the degree of pressure reduction in the solvent removal process fluctuates and becomes higher than the initial setting, the pulling pressure decreases and the supply speed of the W1 / O / W2 emulsion is affected. Loosen to increase supply and maintain supply rate.

(Solvent removal step)
The solvent removal step removes the organic solvent of the oil phase (O) from the W1 / O / W2 emulsion prepared by the secondary emulsification step, and has a lipid bilayer membrane composed of mixed lipid components (F1) and (F2) This is a step of forming liposomes. As the removal of the organic solvent progresses, the hydration of the lipids that make up the liposomes progresses, the multivesicular liposomes dissolve and spread into the single-vesicle liposome state, or from the position close to the interface of the W1 / O / W2 emulsion, It is thought that liposomes are formed by tearing. In this solvent removal step, liposomes are continuously formed while receiving the continuous supply of the W1 / O / W2 emulsion prepared in the secondary emulsification step.

  As one of the methods for continuously producing liposomes in the solvent removal step of the present invention, the W1 / O / W2 emulsion is introduced into the solvent removal flow path, and the organic solvent is evaporated while flowing down the solvent removal flow. An embodiment is preferred in which liposomes are produced before reaching the downstream end of the road. By placing the W1 / O / W2 emulsion in a flow state instead of a stationary state, contact and aggregation between droplets can be reduced, and formation of aggregates such as multivesicular liposomes can be suppressed, and a liposome dispersion Can be recovered continuously.

From the viewpoint of efficiency as described below, in the present invention, by continuously performing the solvent removal step under reduced pressure, the W1 / O / W2 emulsion prepared in the secondary emulsification step can be continuously supplied to the solvent removal step. It is desirable to be done. The solvent removal step is preferably performed under reduced pressure in order to promote the evaporation of the organic solvent. However, this reduced pressure also acts as a pulling pressure for drawing the W1 / O / W2 emulsion. It is possible to receive the supply of the W1 / O / W2 emulsion without using a means for feeding the liquid. Furthermore, the action of such pressure reduction can also generate a flow from the dispersed phase of the W1 / O emulsion to the continuous phase in the secondary emulsification part, from the dispersed phase to the continuous phase, which has been performed by the conventional membrane emulsification method. It is also possible to eliminate or reduce the applied pressure.

  The depressurization condition in the solvent removal step depends on the type of organic solvent used, and the solvent saturation takes into account various circumstances such as the solvent removal rate (required time for the solvent removal step) and the W1 / O / W2 emulsion supply rate. Although it can be suitably adjusted within the range of vapor pressure to atmospheric pressure, it is preferably set within the range of + 1% to 10% of the saturated vapor pressure of the solvent, and is more saturated when mixed with different solvents. It is preferable to match with a solvent species having a high vapor pressure. If the pressure is excessively reduced, the organic solvent is repelled and the droplets are destroyed, which may deteriorate the quality of the liposome. For example, when the organic solvent is mainly composed of hexane, the pressure in the chamber is preferably reduced to 750 to 180 mmHg.

  Moreover, in order to further accelerate the removal of the organic solvent, heating may be used in combination. The temperature condition may be set in a range in which the solvent does not suddenly boil while considering the decompression condition, and in a range in which the inner aqueous phase (W1) does not freeze. For example, a range of 0 to 60 ° C. is preferable. 0 to 25 ° C. is more preferable. When using a heat-sensitive chemical, it is preferable to distill off the solvent at a lower temperature and under reduced pressure conditions. When heating is used in combination, the solvent removal flow path may be heated with a heater.

(Other processes)
As other steps used as necessary, the particle size of the liposome is adjusted to a desired range (for example, about 50 to 500 nm), and the multivesicular liposome formed secondary from the W1 / O / W2 emulsion is separated. It is also used for the production of conventional liposomes, such as a sizing process that can be converted into mononuclear liposomes and a dry powdering process for making liposomes into a form suitable for storage (redispersion in an aqueous solvent at the time of use). Various processes that have been performed are listed. These steps may be provided after the solvent removal step and continuously performed after the solvent removal step.

  Moreover, when using the specific dispersion stabilizer mentioned later in a secondary emulsification process, you may provide the isolation | separation process for isolate | separating a specific dispersion stabilizer and a liposome and removing a specific stable dispersant from a liposome dispersion liquid. . For example, if a microfiltration membrane (MF membrane, pore size of about 50 nm to 10 μm) or an ultrafiltration membrane (UF membrane, pore size of about 2 to 200 nm) is used, a molecular assembly of liposomes and self (for example, a volume average particle size of 10 nm or less) Can be efficiently separated from the specific stable dispersant formed. In view of the use of the product, if there is no problem even if the specific stable dispersant and the liposome are not separated, this separation step may not be provided.

-Liposome production equipment-
The liposome production apparatus of the present invention comprises means for carrying out the production method of the present invention as described above. Hereinafter, the liposome production apparatus 1 will be described with reference to FIG.

(Primary emulsification part)
The primary emulsifying unit 10 for emulsifying the inner aqueous phase (W1) and the oil phase (O) using the mixed lipid component (F1) for liposomes to prepare a W1 / O emulsion is a known emulsifying device / equipment. Other suitable means can be used. For example, in the case of emulsification by the stirring emulsification method mentioned as a preferred method in the primary emulsification step, an aqueous solvent or aqueous solution forming an inner aqueous phase, an organic solvent or organic solution forming an oil phase, and a mixed lipid component ( A primary emulsification part can be comprised by a stirrer (not shown) like a homogenizer which emulsifies these raw materials and a raw material. Examples of the stirrer include a high-speed stirrer (Excel-Auto HOMOGENIZER Nippon Seiki, disk turbine blade), high-pressure homogenizer (Nanomizer NM2-L200AR-D Yoshida Machine Industries), premix membrane emulsification (SPG inline mixer IM-125 SPG Techno) Can be used.

  In addition, a storage container (not shown) and a supply path (not shown) for supplying the respective raw materials of the W1 / O emulsion to the primary emulsification part may be connected to the upstream side of the primary emulsification part. The supply path may include supply control means (not shown) such as a pump and a valve as necessary.

(Secondary emulsification part)
The secondary emulsification part 20 for emulsifying the W1 / O emulsion and the external aqueous phase (W2) prepared in the primary emulsification part using the mixed lipid component (F2) for liposome, It can be constructed using a known device / equipment for membrane emulsification and other appropriate means. For example, in the case of emulsification by the membrane emulsification method (microchannel emulsification method or emulsification method using an SPG membrane) mentioned as a preferred method used in the secondary emulsification step, as shown in FIG. 1, an opening (not shown) The dispersed phase 20a to which the W / O emulsion is fed from, and the emulsified film 20b which is located at the boundary between the dispersed phase 20a and the continuous phase 20c and has pores for forming droplets when the W / O emulsion passes through (Microchannel or SPG film) and an external water phase (W2) is supplied from an opening (not shown) at one end and droplets formed by membrane emulsification from the opening (not shown) at the other end. The secondary emulsification part 20 can be comprised by the continuous phase 20c in which the flow of the external water phase from which the external water phase (namely, W1 / O / W2 emulsion) containing it discharges | emits is discharged | emitted. As such a secondary emulsification part, for example, products such as microchannel emulsification (EP3-0610 EPTEC) and SPG membrane emulsification (high speed mini kit KH-125 SPG Techno) can be used.

  The configuration of the constituent members of the secondary emulsification unit 20 (the terrace length of the microchannel substrate, the channel depth and the channel width, or the pore diameter of the SPG film, etc.) depends on the preparation speed of W1 / O / W2 emulsion and the size of the droplets. Appropriate adjustments can be made to ensure appropriateness. When an SPG membrane is used, the pore diameter is usually 0.1 to 100 μm, preferably 0.1 to 5.0 μm.

  A storage container 25 and a supply path for continuously supplying the external water phase (W2) are connected to the upstream side of the continuous phase 20c of the secondary emulsification unit 20. The supply path detects, for example, the degree of pressure reduction of the solvent removal unit 30 and the liquid feeding pressure of the W1 / O / W2 emulsion by a predetermined member (not shown), to the continuous phase 20c of the outer aqueous phase (W2). It is desirable to provide a supply control means 40 such as a flow rate adjusting valve that controls the supply speed of the gas to be constant.

On the other hand, on the downstream side of the continuous phase 20 c of the secondary emulsification unit 20, the prepared W1 / O / W2 emulsion is discharged from the secondary emulsification unit 20 and supplied to the solvent removal unit 30. And a conduit (W1 / O / W2 emulsion conduit) connecting the solvent removal unit 30 and the solvent removal unit 30 are provided. This conduit includes, for example, a member (not shown) for detecting the degree of pressure reduction of the solvent removing unit 30 and the liquid feeding pressure of the W1 / O / W2 emulsion, and the solvent removal of the W1 / O / W2 emulsion based on the detection result. It is desirable to provide a supply control means 40 such as a flow rate adjusting valve including a member (not shown) having a processing function for controlling the supply speed to the unit 30 to be constant. In addition, when supplying W1 / O / W2 emulsion to a solvent removal part using the pressure reduction of a solvent removal part which is mentioned later, means for actively sending liquids, such as a pump, is a W1 / O / W2 emulsion conduit. It is not necessary to provide it in the middle of

(Two-stage emulsification unit)
The primary emulsification part and the secondary emulsification part may be formed by separate units or may be formed by a unit (two-stage emulsification unit) in which both are integrated.

  For example, when the two emulsification methods are different, for example, the primary emulsification step is performed by the stirring emulsification method and the secondary emulsification step is performed by the membrane emulsification method, usually, as shown in FIG. 1 (for the stirring emulsification method) The unit of the primary emulsification unit 10 and the unit of the secondary emulsification unit 20 (for the membrane emulsification method) are independent from each other, and the W1 / O emulsion prepared in the primary emulsification unit 10 is supplied to the secondary emulsification unit 20. A conduit for connecting the two. In this case, W1 / O emulsion supply control means 40 (pump, valve, W1 / O emulsion storage container, etc.) may be provided in the middle of the conduit as required.

  On the other hand, when the two emulsification methods are the same or can be integrated, for example, both the primary emulsification step and the secondary emulsification step are performed by a membrane emulsification method, the microchannel substrate described in Patent Document 1 described above is used. In addition, a mode in which the primary emulsification part and the secondary emulsification part are present in a unit in which the W1 / O emulsion prepared in the primary emulsification process is immediately supplied to the secondary emulsification process can be taken.

(Solvent removal part)
Solvent removal for continuously forming liposomes by removing the organic solvent in the oil phase from W1 / O / W2 emulsion while receiving continuous supply of W1 / O / W2 emulsion prepared in the secondary emulsification unit The unit 30 can be configured by using various known solvent removal (recovery) devices / equipment and other appropriate means. For example, as shown in FIG. 1, the chamber 30a can be decompressed, an organic solvent condensing device for recovering an organic solvent evaporated from the W1 / O / W2 emulsion inside the chamber, a refrigerant circulation device, and the like. The solvent recovery device 30b is installed in a pressure reducing device 30c such as a pump for reducing the pressure inside the chamber and sucking the organic solvent into the solvent recovery device, and is installed in the chamber 30a, and continuously evaporates the organic solvent from the W1 / O / W2 emulsion. In particular, a solvent removal channel 30d for producing liposomes and a liposome dispersion receptor 30e that is installed at the end of the solvent removal channel 30d and collects the liposome dispersion that has finished flowing down the channel are used. Thus, the solvent removal unit 30 can be configured. Note that a solvent removal device in which the chamber 30a and the solvent recovery device 30b as described above are integrally formed may be used.

  As the solvent removal flow path 30d, for example, an open top (groove type) flow path formed of a material such as glass or plastic is preferable. The form of the flow path (cross-sectional shape and size, flow path shape, flow path length, inclination, etc.) is such that the introduced W1 / O / W2 emulsion is a solvent removal flow path while taking into consideration the decompression and heating conditions described later. It can be adjusted as appropriate so that the solvent is removed and liposomes are formed before the flow of 30d is completed. In general, the smaller the cross-sectional size of the solvent removal flow path 30d, the better the heat exchange efficiency, and the organic solvent tends to be efficiently evaporated. For example, a flow path in which the cross-sectional shape is semicircular or rectangular and the W1 / O / W2 emulsion finishes flowing down over 0.5 to 3.0 hours is preferable as the solvent removal flow path 30d of the present invention.

As described above in the description of the production method of the present invention, in the present invention, W1 / O / W2 prepared in the secondary emulsification step using the reduced pressure of the solvent removal unit (without using an active supply means). The emulsion can be continuously supplied to the solution removal unit. Therefore, it is desirable that the solvent removal unit of the present invention includes a decompression device (such as a pump) that can be used for promoting continuous solvent removal and for such continuous supply. Further, if necessary, the solvent removing unit may include a heating means for directly heating the inside of the chamber or the solvent removing flow path.

(Other components)
In the production method of the present invention, for example, a polycarbonate membrane or a cellulose membrane (pore diameter of 0.1 to 0.4 μm) is used as a filter as a means for performing the sizing step mentioned as another step used as necessary. Can be used, such as “Extruder” manufactured by NOF Liposome Co., Ltd. and “Liponizer” manufactured by Nomura Micro Science Co., Ltd. In addition, as a means for performing the dry powdering step, various known drying apparatuses, preferably freeze-drying apparatuses can be used.

− Production raw materials, liposome form and use −
・ Mixed lipid component (F1) ・ (F2)
The mixed lipid component (F1) used in the primary emulsification step mainly constitutes the inner membrane of the lipid bilayer membrane of the liposome, and the mixed lipid component (F2) used in the secondary emulsification step mainly constitutes the outer membrane. The mixed lipid components (F1) and (F2) may have the same composition or different compositions.

  The composition of these mixed lipid components is not particularly limited, and various mixed lipid components used for the production of liposomes can be used. Generally, however, phospholipids (lecithin derived from animals and plants; phosphatidylcholine) , Phosphatidylserine (DPPS), phosphatidylglycerol (DPPG), phosphatidylinositol, phosphatidic acid or their fatty acid esters, glycerophospholipids; sphingophospholipids; derivatives thereof, etc.) and sterols that contribute to stabilization of lipid membranes ( Cholesterol, phytosterol, ergosterol, derivatives of these, etc.), and glycolipids, glycols, aliphatic amines, long-chain fatty acids (oleic acid, stearic acid, palmitic acid, etc.) and other various functions Contains compound to be given It may be. In the present invention, neutral phospholipids such as dipalmitoyl phosphatidylcholine (DPPC) and dioleyl phosphatidylcholine (DOPC) are commonly used as the phospholipid. Further, F2 contains a lipid component necessary for imparting functionality as a DDS, such as PEGylated phospholipid, so that the liposome surface can be efficiently modified. The mixing ratio of the mixed lipid component may be appropriately adjusted according to the application while considering properties such as the stability of the lipid membrane and the behavior of the liposome in vivo.

・ Inner water phase (W1), outer water phase (W2), oil phase (O)
As the inner aqueous phase (W1) and the outer aqueous phase (W2), an aqueous solvent composed of water and other solvent mixed with water as needed, salts / saccharides for adjusting the osmotic pressure as necessary, pH An aqueous solution to which a buffer solution for adjustment and other functional components (for example, a dispersion stabilizer) are added is used. On the other hand, as the oil phase (O), an organic solution in which various functional components are added to an organic solvent (hexane, chloroform, etc.) that is not mixed with an aqueous solvent as required is used. For example, an organic solvent containing hexane as a main component (50% by volume or more) is preferable because the resulting nano-sized W1 / O emulsion has good monodispersibility.

  The pH of the inner aqueous phase (W1) is usually in the range of 3 to 10, and can be adjusted to a preferred range depending on the mixed lipid component. For example, when oleic acid or DPPGs is used as the mixed lipid component, the pH is preferably 6 to 8.5. An appropriate buffer may be used to adjust the pH.

-Substances to be encapsulated In the present invention, substances to be encapsulated in liposomes (collectively referred to as drugs) are not particularly limited, and are known in the fields of pharmaceuticals, cosmetics, foods, etc. depending on the use of liposomes. Various substances can be used.

Examples of water-soluble drugs for medical use include, for example, contrast agents (nonionic iodine compounds for X-ray contrast, complexes composed of gadolinium and chelating agents for MRI contrast), anticancer agents, and the like. (Adriamycin, Viralbicin, Vincristine, Taxol, Cisplatin, Mitomycin, 5-Fluorouracil, Irinotecan, Estrasite, Epirubicin, Carboplatin, Intron, Gemzar, Methotrexate, Cytarabine, Isoborine, Tegafur, Cisplatin, Etoposide, Topocycin, Bispothecin Famide, melphalan, ifosfamide, tespamine, nimustine, ranimustine, dacarbatin, enositabine, fludarabine, pentostatin, cladribine, daunomycin, acne Rubicin, ibirubicin, amrubicin, actinomycin, taxotere, trastuzumab, rituximab, gemtuzumab, lentinan, schizophyllan, interferon, interleukin, asparaginase, phosfestol, busulfan, bortezomib, alimta, bevacizumab, nemarabine, antibacterial Ride antibiotics, ketolide antibiotics, cephalosporin antibiotics, oxacephem antibiotics, penicillin antibiotics, beta-lactamase combinations, aminoglycoside antibiotics, tetracycline antibiotics, fosfomycin antibiotics, carbapenem antibiotics Antibiotics, penem antibiotics), MRSA / VRE / PRSP infection treatment, polyene antifungal, pyrimidine antifungal, azole antifungal, Candin antifungal agent, New quinolone synthetic antibacterial agent, Antioxidant agent, Anti-inflammatory agent, Blood circulation promoter, Whitening agent, Skin roughening agent, Anti-aging agent, Hair growth promoting agent, Moisturizing agent, Hormone agent, Vitamin , Nucleic acids (DNA or RNA sense or antisense strands, plasmids, vectors, mRNA, siRNA, etc.), proteins (enzymes, antibodies, peptides, etc.), vaccine preparations (toxoids such as tetanus, etc. as antigens; diphtheria, Japanese encephalitis, polio, rubella, mumps, hepatitis and other viruses as antigens; DNA or RNA vaccines, etc.), pharmacologically active substances, dyes / fluorescent dyes, chelating agents, stabilizers, preservatives And pharmaceutical aids such as

  As a method of encapsulating water-soluble drugs in liposomes, (i) water-soluble drugs are dissolved or suspended in advance in the inner aqueous phase (W1) of the primary emulsification step, and at the end of the solvent removal step. A method for obtaining a liposome encapsulating the water-soluble drug, and (ii) producing an empty liposome that does not encapsulate the water-soluble drug, and then adding the water-soluble drug to the aqueous dispersion of the empty liposome. There is a method of adding water-soluble drugs to liposomes by adding or stirring water-soluble drugs when adding or stirring empty liposomes once and redispersing them in an aqueous solvent. Can also be used. Also, fat-soluble drugs are dissolved or suspended in advance in the oil phase (O) in the primary emulsification step according to (i) above, and an aqueous dispersion of empty liposomes as in (ii) above. Can be encapsulated in liposomes (in the lipid bilayer membrane) by the method of adding to and stirring.

-Dispersion stabilizer In this invention, you may mix | blend a dispersion stabilizer with the outer water phase of a secondary emulsification process as needed. As a dispersion stabilizer, a conventionally used substance such as sodium caseinate may be used. However, a dispersion stabilizer that does not form a molecular assembly by itself or a molecular assembly by itself is formed, but its volume average particle size is It is preferable to use a dispersion stabilizer having a size of 10 nm or less (hereinafter referred to as “specific dispersion stabilizer”). Such a specific dispersion stabilizer contributes to improvement of the encapsulation rate of drugs and efficient formation of mononuclear liposomes, and can be easily separated and removed from the obtained liposome dispersion.

It should be noted that an outer aqueous phase containing a “dispersant that does not form molecular aggregates by itself” refers to a substance that does not form molecular aggregates (typically micelles) as a dispersant, regardless of the concentration. When blended in the outer water phase, and substances that form molecular aggregates above a certain concentration (typically a surfactant having a critical micelle concentration) do not reach it as a dispersant in the outer water phase. When blended, it refers to both of these cases. In addition, an external aqueous phase containing “a dispersing agent that forms a molecular assembly by itself but has a volume average particle size of 10 nm or less” refers to an outer aqueous phase that can form a molecular assembly but has a volume average particle size of 10 nm or less. When the substance which is is mix | blended with the outer water phase as a dispersing agent is pointed out.

  It is considered that the specific dispersion stabilizer can be roughly classified into two types from the viewpoint of action. One is, like a polysaccharide, distributed in the entire outer aqueous phase (W2) because the orientation to the interface between the primary emulsion (W1 / O) and the outer aqueous phase (W2) is relatively small. / W2 has a function of stabilizing liposomes by preventing them from sticking to each other. The other is the relatively high orientation of the W1 / O / W2 emulsion at the interface, such as proteins and nonionic surfactants, and has the effect of stabilizing by surrounding the emulsion like a protective colloid. Is.

  When W1 / O / W2 coalesces and the particle size increases, the solvent removal by in-liquid drying method etc. becomes non-uniform and the encapsulated drug is likely to leak out. Stabilizers can suppress such destabilization and contribute to the improvement of mononuclear liposome formation efficiency and drug encapsulation rate. In addition, if the specific dispersion stabilizer is oriented at the interface of the W1 / O / W2 emulsion, it becomes easier to dissolve individual liposomes as the liposomes are formed as the solvent is removed. It contributes to the improvement of the inclusion rate.

  As described above, the specific dispersion stabilizer performs a predetermined function during the formation of the liposome, but the mixed lipid component mainly composed of the phospholipid having a hydration ability has the self-organization ability, so that it is dispersed after the liposome formation. Even without the agent, the dispersed state can be maintained.

  Typical examples of the specific dispersion stabilizer include proteins, polysaccharides, and nonionic surfactants, but are not limited thereto, and other dispersion stabilizers having a specific function as the specific dispersion stabilizer can be used. Substances may be used. If the specified dispersion stabilizer is a substance that has been approved as an additive in pharmaceuticals, etc. (it is guaranteed that it will not have a significant effect on the human body even if administered to the body) However, even if a part of them remain in the liposome dispersion, there is no clinical problem.

  Examples of the protein include gelatin (a soluble protein obtained by denaturing collagen by heating) and albumin. Gelatin usually has a molecular weight distribution of several thousand to several million, and preferably has a weight average molecular weight of 5,000 to 100,000, for example. Gelatin commercially available for medical use or food use can be used. Albumin includes egg albumin (molecular weight of about 45,000), serum albumin (molecular weight of about 66000 ... bovine serum albumin), milk albumin (molecular weight of about 14000 ... α-lactalbumin), and the like. Is preferred.

  Examples of the polysaccharide include dextran, starch, glycogen, agarose, pectin, chitosan, sodium carboxymethylcellulose, xanthan gum, locust bean gum, and guar gum. For example, dextran having a weight average molecular weight of 10,000 to 100,000 is preferable.

Examples of the nonionic surfactant include polyalkylene glycol compounds such as “Tween 80” (Tokyo Chemical Industry Co., Ltd., polyoxyethylene sorbitan monooleate, molecular weight 1309.68) and “Pluronic F-68” (BASF, poly Examples thereof include products such as oxyethylene (160) polyoxypropylene (30) glycol, number average molecular weight 9600) and polyethylene glycol having a weight average molecular weight of 1000 to 8000.

  Whether the specific dispersion stabilizer forms a molecular aggregate by itself in the outer aqueous phase, or whether the volume average particle size of the formed molecular aggregate is 10 nm or less (that is, in the outer aqueous phase Whether or not the added substance satisfies the requirements as a specific dispersion stabilizer) is confirmed by, for example, a dynamic light scattering particle size distribution analyzer or a freeze cleaving method using a transmission electron microscope (TEM). Is possible.

  The amount of the specific dispersion stabilizer added to the outer aqueous phase (the concentration of the specific dispersion stabilizer) may be adjusted in an appropriate range depending on the type. When a substance that forms a molecular aggregate (volume average particle diameter exceeding 10 nm) by itself at a certain concentration is added as a specific dispersion stabilizer, the addition amount is adjusted within a range not reaching the concentration. In addition, depending on the type of the specific dispersion stabilizer, if the concentration is too high, measurement by the particle size distribution system may be hindered. Therefore, it is preferable to adjust the concentration within a low range that does not cause such a hindrance.

  Considering that the liposome and the specific dispersion stabilizer are separated by the filtration step, the volume average particle size of the self-assembled molecular aggregate or the aggregate of the specific dispersion stabilizer is 1/10 or less of the volume average particle size of the liposome. Is preferable, and 1/100 or less is more preferable.

  If the molecular weight of the specific dispersion stabilizer is too small, it tends to be mixed into the lipid membrane and inhibits the formation of liposomes. Conversely, if the molecular weight is too large, the dispersion of the W1 / O / W2 emulsion in the outer aqueous phase may occur. There is a possibility that the orientation speed to the interface is delayed, leading to the coalescence of liposomes and the formation of multivesicular liposomes. Therefore, it is preferable that the weight average molecular weight of a specific dispersion stabilizer exists in the range of 1,000-100,000.

-Form and use of liposomes The form and use of liposomes finally produced by the production method or production apparatus of the present invention (a sizing step or a sizing apparatus may be used if necessary) are not particularly limited. However, for example, when used as a liposome preparation for medical use, the volume average particle diameter is preferably 50 to 1,000 nm, more preferably 50 to 300 nm. Liposomes with such a size have little risk of occluding capillaries and can pass through gaps formed in blood vessels in the vicinity of cancer tissue, so they are convenient for use by being administered to the human body as pharmaceuticals. is there.

(Measuring method of particle size distribution)
The average particle size and CV value of the W1 / O emulsions obtained in the primary emulsification step of Examples and Comparative Examples described below were measured according to the following methods.

  The W1 / O emulsion was diluted 10 times with a dichloromethane / hexane mixed solvent (volume ratio: 4/6, the same specific gravity as the inner aqueous phase), and a dynamic light scattering nanotrack particle size analyzer (UPA-EX150, Nikkiso) Was used to measure the particle size distribution, and based on this, the average particle size and CV value (= (standard deviation / average particle size) × 100 [%]) were calculated.

Example 1
(Production of W1 / O emulsion by primary emulsification process)
15 ml of hexane containing 0.3 g of egg yolk lecithin “COATSOME NC-50” (NOF Corporation) having a phosphatidylcholine content of 95%, 0.152 g of cholesterol (Chol) and 0.108 g of oleic acid (OA) ( O) and 5 mL of tris-hydrochloric acid buffer solution (pH 8, 50 mmol / L) containing calcein (0.4 mM) was used as the aqueous dispersion phase (W1) for the inner aqueous phase. These mixed liquids were put into a 50 mL beaker with a cock, and ultrasonic waves were irradiated at 25 ° C. for 15 minutes by an ultrasonic dispersion apparatus (UH-600S, SMT Co., Ltd.) in which a probe having a diameter of 20 mm was set (output 5. 5) An emulsification treatment was performed. When measured according to the above method, the W1 / O emulsion obtained in the primary emulsification step was confirmed to be a monodispersed W / O emulsion having an average particle size of about 220 nm, and the CV value was 33%.

(Production of W1 / O / W2 emulsion by secondary emulsification process and solvent distillation)
Subsequently, the cock of a 50 mL cocked beaker containing the W1 / O emulsion obtained in the primary emulsification step is opened, and the experimental crossflow type microchannel emulsifier module connected by a medical extension tube X2-25 is opened. The obtained dispersed phase was set so as to be transferred. The line connected to the module outlet is filled with the mobile phase (external water phase), the mobile phase is supplied from the inlet of the line, the outlet of the line is connected with a suction bell-like container for Kiriyama funnel, and the tip of the tube is connected It was introduced into a suction bell-like container and connected to a spiral inclined slider-like channel. The inside of the container is depressurized by a decompression device (diaphragm pump: DIVAC 0.6L, Tokyo Rika Machine) connected to a cock attached to the top of the suction bell-like container, the dispersed phase passes through the microchannel, and the mobile phase sucks The valve was adjusted to be sucked into the bell-like container. The W1 / O / W2 emulsion generated in the mobile phase line is introduced into a suction bell-like container, passes through the slider-like channel, and takes 15 minutes to fall into the recovery bottle. The length and inclination of the slider-like flow path were set in the above, and the liposome suspension was recovered in a recovery bottle. The degree of vacuum at this time was set to 750 mmHg, and after reducing the pressure for 3 hours, the pressure reduction was released and the recovery bottle was taken out. As a result, a suspension of fine liposome particles was obtained, and it was confirmed that calcein was contained in the particles.

  The microchannel substrate of the module was made of silicon, and the terrace length, channel depth and channel width of the microchannel substrate were about 60 μm, about 11 μm and about 16 μm, respectively. A glass plate was pressure-bonded to the microchannel substrate to form a channel, and the outlet side of the channel was filled with a Tris-hydrochloric acid buffer solution (pH 8, 50 mmol / L) as an external aqueous phase solution (W2).

Example 2
Tris-HCl buffer solution (pH 8, 50 mmol / L) containing 3% alkali-treated gelatin (isoelectric point about 5) was added to Tris-HCl buffer solution (W2), which is an outer aqueous phase solution (W2). The liposome suspension was recovered in the same manner as in Example 1 except that

Comparative Example 1
(Production of W1 / O emulsion by primary emulsification process)
A W1 / O emulsion was produced in the same manner as in Example 1.

(Production of W1 / O / W2 emulsion by secondary emulsification process)
Subsequently, the W1 / O emulsion obtained by the primary emulsification step is used as a dispersed phase, and a W1 / O / W2 emulsion is produced by a microchannel emulsification method using a laboratory cross-flow type microchannel emulsifier module. It was.

  The microchannel substrate of the module was made of silicon, and the terrace length, channel depth and channel width of the microchannel substrate were about 60 μm, about 11 μm and about 16 μm, respectively. A glass plate is pressure-bonded to the microchannel substrate to form a channel, and the outlet side of the channel is filled with a Tris-HCl buffer solution (pH 8, 50 mmol / L) that is an external aqueous phase solution (W2). The W1 / O emulsion was supplied from the inlet side to produce a W1 / O / W2 emulsion.

(Production of liposomes by removal of organic solvent phase)
Next, the W1 / O / W2 emulsion was transferred to an open glass container without a lid, and stirred with a stir bar at normal pressure (760 mmHg) at room temperature for about 20 hours to volatilize hexane. A suspension of fine liposome particles was not obtained, and the aggregation state was observed with an optical microscope.

Comparative Example 2
Tris-HCl buffer solution (pH 8, 50 mmol / L) containing 3% alkali-treated gelatin (isoelectric point about 5) was added to Tris-HCl buffer solution (W2), which is an outer aqueous phase solution (W2). A liposome suspension was recovered in the same manner as in Comparative Example 1 except that A suspension of fine liposome particles was obtained, and it was confirmed that calcein was contained in the particles.

(Evaluation of inclusion rate)
The encapsulation rate of the liposomes obtained in the above examples and comparative examples was measured according to the following method (results are shown in Table 1).

The fluorescence intensity (F _total ) of the entire liposome aqueous solution (3 mL) was measured using a spectrophotometer (U-3310).
, JASCO Corporation). Next, 0.01M CoCl ₂ Tris-HCl buffer 3
The fluorescence intensity (F _in ) in the vesicle was measured by quenching the fluorescence of calcein leaked into the outer aqueous phase by adding 0 μL with Co ²⁺ . Furthermore, vesicles were prepared under the same conditions as the sample without adding calcein, and the fluorescence (F ₁ ) emitted by the lipids themselves was measured. The inclusion rate was calculated from the following formula;
Inclusion rate E (%) = (F _in −F _l ) / (F _total −F _l ) × 100
(Method for observing the number of micron-sized aggregated particles)
The number of micron-sized particles remaining in the liposome dispersions obtained in the above examples and comparative examples was evaluated by observing with an optical microscope (results are shown in Table 1).

(Discussion)
In Comparative Example 1, preparation of liposomes was attempted without using a dispersion stabilizer, but the emulsified state in the steps of (W1 / O / W2 emulsion production) and (liposome production by removal of organic solvent phase) was poor, and optical Many large aggregates and aggregates of about 10 μm (multi-encapsulated liposomes) were observed by observation with a microscope. On the other hand, in Comparative Example 2, the emulsified state was improved by using alkali-treated gelatin as the dispersion stabilizer, and no large aggregates were found by observation with an optical microscope, and nanoparticles that could not be confirmed visually were obtained. Compared to Example 2, the CV value was slightly high and the inclusion rate was slightly low.

  In Example 1, the preparation of liposomes was attempted without using a dispersion stabilizer, but the aggregates (multi-encapsulated liposomes) of about 10 μm seen in Comparative Example 1 were only slightly observed with an optical microscope. The emulsified state in the steps of (production of W1 / O / W2 emulsion) and (production of liposomes by removing the organic solvent phase) was good. In Example 2, when a dispersion stabilizer was also added in the flow manufacturing process, the result was the same as in Example 1. From these results, it can be considered that the flow production process can be a practical production method that can produce high-quality liposomes that contain almost no agglomerates, have a low CV value, and a high encapsulation rate, without using a dispersion stabilizer. .

DESCRIPTION OF SYMBOLS 1 Liposome manufacturing apparatus 10 Primary emulsification part 20 Secondary emulsification part 20a Dispersed phase 20b Emulsion film 20c Continuous phase 25 Outer water phase storage container 30 Solvent removal part 30a Chamber 30b Solvent recovery apparatus 30c Depressurization apparatus (pump)
30d Solvent removal flow path 30e Liposome dispersion receiver 40 Supply control means

Claims (18)

A primary emulsification step of emulsifying the inner aqueous phase (W1) and the oil phase (O) with the mixed lipid component for liposome (F1) to prepare a W1 / O emulsion;
A secondary emulsification step of emulsifying the W1 / O emulsion and the external aqueous phase (W2) prepared in the primary emulsification step using the mixed lipid component for liposome (F2) to prepare a W1 / O / W2 emulsion;
A method for producing liposomes comprising a solvent removal step of removing the organic solvent of the oil phase (O) from the W1 / O / W2 emulsion prepared in the secondary emulsification step to form liposomes,
The solvent removal step is a method for continuously producing liposomes by receiving continuous supply of the W1 / O / W2 emulsion prepared in the secondary emulsification step and continuously producing liposomes. Manufacturing method.   The continuous production of liposomes in the solvent removal step introduces the W1 / O / W2 emulsion into the solvent removal channel, evaporates the organic solvent while flowing down the liposome, and reaches the end of the solvent removal channel. The method for producing a liposome according to claim 1, wherein the method is performed by forming.   The continuous supply to the solvent removal process of the W1 / O / W2 emulsion prepared in the secondary emulsification process is performed by performing the solvent removal process under reduced pressure. The manufacturing method of the liposome.   The continuous supply to the solvent removal step of the W1 / O / W2 emulsion prepared in the secondary emulsification step is based on the result of detecting the degree of vacuum in the solvent removal step and the liquid feeding pressure of the W1 / O / W2 emulsion. The method for producing a liposome according to any one of claims 1 to 3, which is controlled so as to be performed at a constant rate.   The manufacturing method of the liposome in any one of Claims 1-4 whose emulsification method in the said secondary emulsification process is a membrane emulsification method.   The method for producing liposomes according to claim 5, wherein the membrane emulsification method is a microchannel emulsification method or an emulsification method using an SPG membrane.   The manufacturing method of the liposome in any one of Claims 1-6 whose emulsification method in the said primary emulsification process is a stirring emulsification method.   The method for producing a liposome according to any one of claims 1 to 7, wherein the solvent removing step is performed under a reduced pressure of 750 to 180 mmHg.   The method for producing a liposome according to any one of claims 1 to 8, wherein the primary emulsification step is further performed after adding a substance to be encapsulated in the liposome.   The manufacturing method of the liposome in any one of Claims 1-9 in which the outer water phase (W2) in the said secondary emulsification process contains a dispersion stabilizer.   11. The dispersion stabilizer is a dispersion stabilizer that does not form a molecular assembly by itself or a dispersion stabilizer that forms a molecular assembly by itself, but the volume average particle size of the molecular assembly is 10 nm or less. The manufacturing method of the liposome as described in above. A primary emulsification part for emulsifying the inner aqueous phase (W1) and the oil phase (O) with the mixed lipid component (F1) for liposomes to prepare a W1 / O emulsion;
A secondary emulsification part for emulsifying the W1 / O emulsion prepared in the primary emulsification part and the outer aqueous phase (W2) using the mixed lipid component for liposome (F2) to prepare a W1 / O / W2 emulsion;
A liposome production apparatus comprising a solvent removal unit that removes the organic solvent of the oil phase (O) from the W1 / O / W2 emulsion prepared in the secondary emulsification unit to form liposomes,
The said solvent removal part receives the continuous supply of the W1 / O / W2 emulsion prepared by the said secondary emulsification part, and manufactures a liposome continuously, The manufacturing apparatus of a liposome characterized by the above-mentioned. .   The liposome production apparatus according to claim 12, further comprising a solvent removal channel for evaporating the organic solvent while introducing and flowing down the W1 / O / W2 emulsion for continuous production of liposomes in the solvent removal unit. .   The said solvent removal part is equipped with a decompression device, The continuous supply of the W1 / O / W2 emulsion prepared in the said secondary emulsification part to the said solvent removal part is performed using the said decompression device. 2. The liposome production apparatus according to 1.   In order to continuously supply the W1 / O / W2 emulsion prepared in the secondary emulsification unit to the solvent removal unit, the degree of vacuum in the solvent removal unit and the liquid feeding pressure of the W1 / O / W2 emulsion are detected. The liposome production apparatus according to any one of claims 12 to 14, further comprising supply control means including a member to be controlled and a member for controlling a supply speed to be constant based on a detection result thereof.   The method for producing a liposome according to any one of claims 12 to 15, wherein the emulsification method in the secondary emulsification part is a membrane emulsification method.   The liposome production apparatus according to claim 16, wherein the membrane emulsification method is a microchannel emulsification method or an emulsification method using an SPG membrane.   The liposome production apparatus according to any one of claims 12 to 17, wherein the emulsification method in the primary emulsification part is a stirring emulsification method.